1. Field of the Invention
The present invention relates to improvements in methods and apparatus for selectively extracting cells of interest from a whole blood sample. More particularly, it relates to a readily automatable method for separating and harvesting blood cells on the basis of their respective densities.
2. The Prior Art
A detailed characterization of different types of cells, e.g., lymphocytes, monocytes, eosinophils, etc., found in a whole blood sample can provide a phenotypic and functional fingerprint of a patient's immune system at different and various levels of activation. This information is vital to various investigative strategies, e.g., in the pharmaceutical industry, to determine a new drug's efficacy and toxicity. For this purpose, pharmaceutical companies conduct large clinical trials that include multiple sample collection and test sites throughout the world where cells of interest are separated from other cells and harvested for investigation.
To date, the process for preparing blood samples for buoyant density cell separation has been tedious and labor-intensive, as has been the cell-harvesting process itself. The large number of samples and differences in test site practices creates, in general, a difficult challenge for data interpretation and generation of technical conclusions.
Heretofore, it has been common to manually prepare blood samples for mononuclear cell separation by first carefully layering the blood sample atop the surface of a suitable density gradient medium disposed within a centrifugation tube. During this layering process, should any turbulence be created at the sample/density gradient interface, an undetermined number of blood cells will become “lost” within the density gradient material. After layering the blood sample atop the density gradient material, the tube and its contents are subjected to a relatively low speed (e.g., 200 to 400 g-force) centrifugation for a short time (e.g., about 30 minutes). This centrifugation step causes differential movement of the different blood cell types within the tube until all of the cells reach a buoyant equilibrium; at this time, the relatively dense granulocytes and erythrocytes will have moved to the bottom of the tube and formed a solid pellet, the platelets and plasma will have moved to the top portion of the tube, above the density gradient material, and the targeted mononuclear cells will have formed a distinct relatively thin layer, e.g., about 1.5 mm thick, located at the plasma/density gradient interface. Harvesting of the mononuclear cells is then achieved by manually lowering a pipette into the mononuclear cell layer and drawing out a desired volume of cells from this layer. While, in principle, this method is relatively simple to perform, achieving uniformity of results, from one person to the next, as well as from one laboratory to the next, is difficult, at best.
As indicated above, it is recognized that the above process for layering a blood sample atop a density gradient material is a relatively tedious and time-consuming process. A technique that is intended to address these issues is to use a hand-held and hand-operated dispenser that is adapted to inject the density gradient material underneath a blood sample in a tube, rather than to carefully add the blood sample to the top surface of a density gradient layer already in the tube. Such dispenser comprises a narrow dispensing probe that is suitably connected to a hand-held syringe containing the density gradient material. In use, the dispensing probe is manually inserted inside a blood sample-containing tube and advanced through the blood sample to a position proximate the bottom of the tube. The syringe is then activated to slowly inject the density gradient material at the tube bottom, thereby causing the blood sample to rise above it. This so-called “underlaying” technique, and a suitable dispenser construction for carrying it out, are reported in a Technical Report by A. Islam, appearing in Journal of Clinical Pathology, 1995, Vol. 48, pp. 686-688.
While using a density gradient dispenser of the type discussed above theoretically affords certain advantages over the more conventional overlayering technique for disposing a blood sample atop a density gradient material, the use of such a device does not necessarily produce uniform results. Unless the density gradient medium is continually injected at an optimum location at the bottom of the tube, and injected at a relatively steady flow rate, a turbulent disturbance may occur with an unpredictable number of cells from the sample becoming trapped in the density gradient material, and most of those cells will be unavailable for subsequent harvesting. Clearly, there is a need to remove this manual involvement in the cell separation process in order to reduce the variability in the number of cells eventually harvested.